Isothiazoloquinolones containing functionalized aromatic hydrocarbons at the 7-position: Synthesis and in vitro activity of a series of potent antibacterial agents with diminished cytotoxicity in human cells

Article abstract of DOI:10.1016/j.bmcl.2005.11.065

This report describes 9H-isothiazolo[5,4-b]quinoline-3,4-diones (ITQs) containing aromatic groups at the 7-position that were prepared using palladium-catalyzed cross-coupling and tested against a panel of susceptible and resistant bacteria. In general, these compounds were more effective against Gram-positive than Gram-negative organisms. Many of the ITQs were more potent than contemporary quinolones and displayed a particularly strong antistaphylococcal activity against a clinically important, multi-drug-resistant strain. In contrast with ITQs reported previously, several of the analogues described in this Letter demonstrated low cytotoxic activity against a human cell line.

Full text of DOI:10.1016/j.bmcl.2005.11.065

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1272–1276
Isothiazoloquinolones containing functionalized aromatic
hydrocarbons at the 7-position: Synthesis and in vitro
activity of a series of potent antibacterial agents with
diminished cytotoxicity in human cells
Jason A. Wiles,* Qiuping Wang, Edlaine Lucien, Akihiro Hashimoto, Yongsheng Song,
Jijun Cheng, Christopher W. Marlor, Yangsi Ou, Steven D. Podos, Jane A. Thanassi,
Christy L. Thoma, Milind Deshpande, Michael J. Pucci and Barton J. Bradbury*
Achillion Pharmaceuticals, Inc., 300 George Street, New Haven, CT 06511-6653, USA
Received 27 October 2005; accepted 18 November 2005
Available online 7 December 2005
Abstract—This report describes 9H-isothiazolo[5,4-b]quinoline-3,4-diones (ITQs) containing aromatic groups at the 7-position that
were prepared using palladium-catalyzed cross-coupling and tested against a panel of susceptible and resistant bacteria. In general,
these compounds were more effective against Gram-positive than Gram-negative organisms. Many of the ITQs were more potent
than contemporary quinolones and displayed a particularly strong antistaphylococcal activity against a clinically important, multi-
drug-resistant strain. In contrast with ITQs reported previously, several of the analogues described in this Letter demonstrated low
cytotoxic activity against a human cell line.
ꢀ 2005 Elsevier Ltd. All rights reserved.
The quinolones are a well-known class of broad-spec-
trum bactericidal agents that inhibit essential type II
bacterial topoisomerases such as DNA gyrase1,2 and
topoisomerase IV.3 Although several generations of
quinolones have emerged in the 40 years since their
initial discovery, the core structure of therapeutically
useful quinolones continues to incorporate the 4-pyri-
done-3-carboxylic acid moiety (Fig. 1).4 Bioisosteric
substitution of the carboxylic acid at C-3 is generally
detrimental to the antibacterial activity of quinolones;5
however, replacement of this group with a ring-fused
isothiazolone surrogate enhanced bioactivity (2- to 10-
fold based on limited published data) of the resulting
isothiazoloquinolones (ITQs, Fig. 1).6–9 These tricyclic
quinolones utilized small nitrogen-containing heterocy-
cles attached directly to the ITQ nucleus at C-7 through
a C–N bond (nitrogen-coupled). Unfortunately, these
potent antibacterial agents inhibited both bacterial and
O
O
O
O
3
5
8
5
8
4
4
F
6
F
6
OH
3
NH
7
7
2
2
S
N
N9
N
N1
1
HN
HN
quinolone
(4-pyridone-3-carboxylic acid)
isothiazoloquinolone
(ITQ)
Figure 1. Structures and numbering schemes of a quinolone (cipro-
floxacin) and an isothiazoloquinolone (A-62824).
mammalian topoisomerase II10 and caused related topo-
isomerase II-mediated DNA breakage in cell-free as-
says—an activity that was correlated with mammalian
cytotoxicity.11 Another successful approach for prepar-
ing potent analogues with structures that deviate from
conventional quinolones is replacement of the amino
substituents at C-7—groups studied extensively because
of their strong influence on potency and safety of quin-
olones12—with aromatic groups bonded directly to the
quinolone nucleus through a C–C bond (carbon-cou-
pled).13,14 Garenoxacin, a quinolone of this subclass that
was described recently,15–17 has shown great promise in
advancing toward the market due, in part, to its strong
Keywords: Antibacterial; Methicillin-resistant Staphylococcus aureus;
Quinolone.
*
Corresponding authors. Tel.: +1 203 624 7000; fax: +1 203 752 5454
(J.A.W); e-mail addresses: jwiles@achillion.com; bbradbury@
achillion.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.065

J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1272–1276
1273
activity against quinolone-resistant Gram-positive
strains. As part of our efforts to develop new quinolones
with exceptional potency against emerging quinolone-
resistant organisms, we describe the synthesis and anti-
bacterial evaluation of novel ITQs that contain aromatic
groups carbon-coupled to the 7-position. Specifically,
we limit our discussion to analogues that contain func-
tionalized phenyl substituents. Our primary objectives
are (a) to optimize the potency of these compounds
against resistant strains of Staphylococcus aureus and
(b) to restrict their mammalian cytotoxicity to concen-
trations that exceed their therapeutic range.
group with sodium hydrosulfide was unnecessary: meth-
yl thioether 3 was converted directly to thiol 4 in 84%
yield using excess anhydrous sodium hydrosulfide with
gentle heating in tetrahydrofuran. To prevent oxidative
degradation of 4, we reacted this compound directly
(without purification) with hydroxylamine-O-sulfonic
acid under basic conditions to give the desired ITQ
intermediate 519 in 85% yield. Microwave-assisted Suzu-
ki–Miyaura cross-coupling of the ITQ nucleus 5 with
the desired boronic acids or dioxaborolanes furnished
derivatives 6–38, typically, in 30–50% yield after HPLC
purification,20 with 2-substituted phenyl derivatives hav-
ing the lowest yields. The boronic acids and dioxaborol-
anes used in this study were either (a) purchased from
commercial suppliers or (b) prepared from the corre-
sponding commercially available bromides using stan-
dard methods.21 The exceptions were boronic acid 40
and dioxaborolane 42 (Scheme 2) that were used to pre-
pare ITQ analogues 35 and 36, respectively.
Our synthetic strategy involved introduction of carbon-
linked pendant groups via palladium-catalyzed cross-
coupling of organoboron compounds with an ITQ
nucleus containing a bromide at C-7. The 7-bromo
ITQ nucleus was prepared from keto ester 114 using
the synthetic sequence outlined in Scheme 1 that fol-
lowed an earlier report on the synthesis of the 7-fluoro
ITQ nucleus.18 Sequential treatment of 1 with cyclopro-
pyl isothiocyanate, sodium hydride, and methyl iodide
at room temperature generated ketene N,S-acetal 2 in
94% yield. Subsequent reaction of 2 with sodium hy-
dride at 75 ꢁC effected ring closure to give 3 in 93% yield.
Unlike that reported for the 7-fluoro ITQ nucleus, we
found that oxidation of 3 with m-chloroperbenzoic acid
followed by displacement of the resulting methyl sulfinyl
The minimum inhibitory concentrations (MICs)22 of
ITQ analogues were determined against quinolone-sus-
ceptible and quinolone-resistant pathogens (Tables 1
and 2), and were compared directly with that of their
corresponding 3-carboxylic acid counterpart ciprofloxa-
cin (CIP). Because this class of compounds was also ex-
plored previously as antineoplastic agents,11 we tested
their cytotoxic activity in human Hep2 cells (laryngeal
carcinoma).23 Most of the ITQs were more effective
against Gram-positive (S. aureus) than Gram-negative
(Escherichia coli) organisms. Several ITQs were excep-
tionally potent (MICs 61 lg/mL) against methicillin-re-
sistant S. aureus (MRSA, Table 1)—activities that were
superior to those of contemporary quinolones such as
gatifloxacin (GAT), gemifloxacin (GEM), and moxiflox-
acin (MFX). One of the most active compounds that we
tested against MRSA was the hydroxy analogue 18. This
compound, however, showed strong inhibitory activity
against human topoisomerase II24 (EC2 = 15 lM25)
and strong cytotoxic activity (Table 1). Nitrogen-cou-
pled ITQs described previously10,11 (e.g., A-62824,
Fig. 1) also showed strong activity against mammalian
topoisomerase II (EC2 = 20 lM) and strong cytotoxic
activity (Table 1). Our data suggest that this undesirable
activity may not be a class effect for ITQs,26 but rather, a
complex function that relies heavily on the contributions
of the individual C-7 components. For example, as
O
O
O
F
O
OEt
F
F
a
OEt
SMe
F HN
Br
Br
1
2
b
O
O
O
O
F
F
c
OEt
SMe
OEt
SH
Br
N
Br
N
3
4
d
O
O
OH
O
O
NH
OH
a
F
F
B
B
e
OH
OH
NH
N
C
S
S
Ar
N
Br
N
H2N
39
40
6−38
5
O
B
O
B
b, c
Scheme 1. Reagents and conditions: (a) c-PrNCS (1.7 equiv), DMF,
rt, then NaH (1.07 equiv), 0 ꢁC ! rt, 20 h, then MeI (1.7 equiv), rt, 4 h,
94%; (b) NaH (1.05 equiv), DMF, 75 ꢁC, 18 h, 93%; (c) anhydrous
NaSH (1.5 equiv), THF, 45 ꢁC, 18 h, 84%; (d) H2NOSO3H (4.2 equiv),
NaHCO3 (10 equiv), H2O/THF, 85%; (e) ArB(ORÕ)2 where Ar = aryl
and R0 = H or –C(CH3)2– (2–3 equiv), NaHCO3 (10 equiv), Pd(PPh3)4
(5 mol %), DMF/H2O, MWI (130 ꢁC), 10–20 min, 30–50% yield after
HPLC purification.
O
O
O
H2N
N
H2N
H
41
42
Scheme 2. Reagents and conditions: (a) H2 (3 atm), 10% Pd/C, EtOH,
rt, 48 h, quant.; (b) Boc-glycine (1.5 equiv), BOP (1.5 equiv), DIEA
(4.2 equiv), DMF, rt, 3 h, 70%; (c) neat TFA, rt, 1 h, quant.

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J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1272–1276
Table 1. Minimum inhibitory concentrations (MICs) and cytotoxic activities of ITQsa
O
O
F
NH
R
S
N
Compound
R
MIC (lg/mL)
Sa
Cytotox (72 h) CC50 (lM)
Ec
MRSA (MRQRVI)
CIP
A-62824
GAT
GEM
MFX
6
0.02
0.25
0.05
32
>100
7
67
0.004
0.015
0.015
0.015
0.06
4.0
8.0
2.0
2.0
16
0.125
0.03
0.06
46
>100
>100
ND
>100
ND
>85
>100
>100
84
H
0.004
0.008
0.004
0.004
0.125
0.001
0.03
7
3-H2NC(O)
3-Ac
0.125
0.125
0.125
2.0
2.0
2.0
64
8
9
10
3-AcNH
2-NC
3-NC
>64
0.125
16
11
12
0.03
1.0
4-NC
13
14
4-NC-CH2
2-F
3-F
0.02
0.002
0.015
0.004
0.03
0.50
8.0
1.0
32
0.125
0.125
0.25
>100
96
15
16
4-F
3-HO
>100
>100
8
17
18
0.06
0.03
0.004
0.004
0.125
0.004
0.015
0.001
0.001
0.008
0.004
0.03
0.25
0.125
32
4-HO
2-MeO
19
20
2.0
>84
26
3-MeO
4-MeO
0.125
0.125
0.06
2.0
2.0
0.125
2.0
2.0
4.0
64
21
22
91
4
3-MeO-4-HO
4-HO-3,5-Me2
3-HO-CH2
4-HO-CH2
2-H2N
23
24
0.125
0.125
0.06
ND
ND
25
25
26
0.125
0.004
0.06
46
27
28
3-H2N
4-H2N
0.008
0.004
0.008
0.008
0.06
8.0
0.50
2.0
0.50
32
>100
34
18
29
30
3-H2N-4-Me
3-H2N-4-F
3-Me2N
0.125
0.125
2.0
28
>100
51
31
32
4-Me2N
0.50
0.001
0.015
0.03
4.0
2.0
8.0
>64
64
33
3-H2N-CH2
4-H2N-CH2
4-H2N(CH2)2
4-H2NCH2C(O)NH
3-(2-Piperidinyl)
4-(2-Piperidinyl)
0.015
0.015
0.06
8
34
4
11
35b
36b
37c
38c
0.02
0.25
1.0
0.125
0.25
>75
4
0.06
0.125
8.0
4.0
4
a Abbreviations: CIP, ciprofloxacin; Ec, Escherichia coli ATCC 25922; GAT, gatifloxacin; GEM, gemifloxacin; MFX, moxifloxacin; MRQRVI,
methicillin- and quinolone-resistant, vancomycin intermediate-resistant; MRSA, methicillin-resistant Staphylococcus aureus ATCC 700699; ND,
not determined; Sa, Staphylococcus aureus ATCC 29213.
b Prepared from the custom-made boronic acid or dioxaborolane (Scheme 2).
c Prepared from the custom-made dioxaborolane derived from the commercially available bromide.
shown previously for the quinolones,24 transposition of
the 4-hydroxy group of 18 to the 3-position (17) strongly
diminished the cytotoxic activity. Several other ITQs
listed in Table 1 also exhibited low cytotoxicity (CC50
>100 lM). Compound 15 also demonstrates that both
strong cytotoxicity and inhibition of human topoisomer-
ase II are not class effects for all ITQs: 15 exhibited a 4-
to 12-fold better activity (MICs) against S. aureus
strains and >7-fold less cytotoxic and human topoiso-
merase II activity than A-62824 (15, CC50 = 96 lM
and EC2 >150 lM). In addition, ITQs were active
against the target enzymes, inhibiting gyrase (E. coli)
and topoisomerase IV (S. aureus) at levels comparable
with those of quinolones (Table 2).27
In general, substitution at the 2-position of the C-7
phenyl group effected significant reduction in antibacte-
rial activity (increase in MIC) when compared directly
with those of the 3- and 4-substituted counterparts (Ta-
ble 1). For most substituents, the activity of 3-substitut-
ed analogues against MRSA was greater than that of the
corresponding 4-substituted analogues (e.g., cf. 11 and

J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1272–1276
1275
Table 2. Minimum inhibitory concentrations (MICs) and activities
against the target enzymes gyrase and topoisomerase IV of selected
ITQsa
6. Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Shen,
L.; Pernet, A. G. Drugs Exp. Clin. Res. 1988, 14, 379.
7. Chu, D. T. U.S. Patent 4,767,762, 1988.
8. Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Shen,
L.; Pernet, A. G. In Quinolones. Proceedings of an
International Telesymposium; Fernandes, P. B., Eds.;
Prous Science Publishers: Barcelona, Spain, 1989.
9. Chu, D. T. W.; Lico, I. M.; Claiborne, A. K.; Plattner, J.
J.; Pernet, A. G. Drugs Exp. Clin. Res. 1990, 16, 215.
10. Kohlbrenner, W. E.; Wideburg, N.; Weigl, D.; Saldivar,
A.; Chu, D. T. W. Antimicrob. Agents Chemother. 1992,
36, 81.
11. Chu, D. T.-W.; Plattner, J. J.; Shen, L. L.; Klein, L. L.
U.S. Patent 5,071,848, 1991.
12. Domagala, J. M. J. Antimicrob. Chemother. 1994, 33, 685.
13. Jones, R. N.; Barry, A. L. Antimicrob. Agents Chemother.
1990, 34, 306.
14. Reuman, M.; Daum, S. J.; Singh, B.; Wentland, M. P.;
Perni, R. B.; Pennock, P.; Carabateas, P. M.; Gruett, M.
D.; Saindane, M. T.; Dorff, P. H.; Coughlin, S. A.;
Sedlock, D. M.; Rake, J. B.; Lesher, G. Y. J. Med. Chem.
1995, 38, 2531.
15. Lawrence, L. E.; Wu, P.; Fan, L.; Gouveia, K. E.; Card,
A.; Casperson, M.; Denbleyker, K.; Barrett, J. F. J.
Antimicrob. Chemother. 2001, 48, 195.
16. Wu, P.; Lawrence, L. E.; Denbleyker, K. L.; Barrett, J. F.
Antimicrob. Agents Chemother. 2001, 45, 3660.
17. Hayashi, K.; Takahata, M.; Kawamura, Y.; Todo, Y.
Arzneim-Forsch/Drug Res. 2002, 52, 903.
Compound
Ec MIC
(lg/mL)
Gyrase
(WT Ec)
IC50 (lM)
Sa MIC
(lg/mL)
Topo IV
(WT Sa)
IC50 (lM)
CIP
A-62824
GAT
GEM
MFX
15
0.02
0.3
0.1
0.5
0.5
0.3
1.0
0.2
0.8
0.5
0.9
3.1
0.3
0.3
0.1
0.1
0.7
1.0
0.25
0.05
1.0
0.5
1.2
0.3
0.8
7.2
1.6
25
0.004
0.015
0.015
0.015
0.125
0.06
0.125
0.03
0.06
0.004
0.004
0.004
0.008
0.004
0.008
0.004
0.008
0.015
0.03
17
18
0.03
0.125
0.06
24
25
8.2
41
27
28
0.004
0.06
10
12
30
33
0.125
0.015
0.015
0.125
0.25
10
0.2
1.5
2.7
2.6
34
37
0.06
0.125
38
a Abbreviations: CIP, ciprofloxacin; Ec, Escherichia coli ATCC 25922;
GAT, gatifloxacin; GEM, gemifloxacin; MFX, moxifloxacin; ND,
not determined; WT, wild-type; Sa, Staphylococcus aureus ATCC
29213.
18. Chu, D. T. W. J. Heterocycl. Chem. 1990, 27, 839.
15 with 12 and 16, respectively). In many instances,
inclusion of OH or NH2 into the C-7 phenyl group also
imparted strong activity against susceptible E. coli (Ta-
ble 2). Further modification of the OH and NH2 groups
(e.g., one-carbon homologation and methylation) yield-
ed analogues with reduced overall antibacterial activity.
1
19. 5: H NMR (300 MHz, DMF-d7, 60 ꢁC): d 1.36 (m, 2H),
1.48 (m, 2H), 3.70 (m, 1H), 8.04 (d, JH-F = 8.5 Hz, 1H),
8.45 (d, JH-F = 5.5 Hz, 1H). 13C{1H} NMR (75 MHz,
DMF-d7, 60 ꢁC):
d
9.5, 32.7, 108.0, 112.3 (d,
JC-F = 24.0 Hz), 115.3 (d, JC-F = 23.0 Hz), 123.0, 126.6
(d, JC-F = 5.5 Hz), 140.1 (d, JC-F = 2.0 Hz), 155.7 (d,
JC-F = 244.5 Hz), 165.3, 170.4, 172.6 (d, JC-F = 2.0 Hz).
19F{1H} NMR (282 MHz, DMF-d7, 60 ꢁC): d ꢁ115.4 (s).
HRMS m/z calcd for C13H879BrFN2NaO2S 376.9372
([M+Na]+); found 376.9367. Anal. Calcd for
C13H8BrFN2O2S: C, 43.96; H, 2.27; N, 7.89. Found: C,
43.73; H, 2.50; N, 7.54.
In summary, we described novel carbon-coupled ITQs
that displayed potent antistaphylococcal activities
(MICs ꢀ0.1 lg/mL against MRSA) that are superior
to those of ciprofloxacin, gatifloxacin, gemifloxacin,
and moxifloxacin. Several of these ITQs exhibited cyto-
toxicity at concentrations (CC50 >100 lM) well above
their projected therapeutic range. Unlike data for previ-
ously reported compounds of this class, our data suggest
that high mammalian cytotoxicity is not a class effect of
ITQs and can be attenuated by judicious choice of sub-
stituents placed at the 7-position. A small set of com-
pounds (11, 13, 15, and 17) displayed acceptable levels
of cellular toxicity (CC50 >80 lM) and in vitro activity
against MRSA (61 lg/mL) that are necessary for con-
sideration as candidates for further in vitro and in vivo
profiling.
20. General procedure: Under an atmosphere of argon, a
reaction vessel was charged with 7-bromo-9-cyclopropyl-
8-methoxy-9H-isothiazolo[5,4-b]quinoline-3,4-dione (0.1 mmol),
dimethylformamide
(4 mL),
tetrakis(triphenylphos-
phine)palladium(0) (0.005 mmol), the desired boronic acid
or dioxaborolane (0.2–0.3 mmol), and a 1 M aqueous
solution of sodium bicarbonate (1 mmol). The resulting
mixture was irradiated with microwaves (CEM Discover)
at 130 ꢁC for 10–20 min, allowed to cool, and evaporated
to dryness under reduced pressure. The isolated residues
were purified using preparative HPLC to give the desired
products. Preparative HPLC was performed using a YMC
Pack Pro C18 150 · 20.0 mm · 5 lm column with an
isocratic elution of 0.35 min at 90:10 H2O/CH3CN con-
taining 0.1% TFA followed by a 23-min linear gradient
elution from 90:10 to 10:90 at a flow rate of 18.9 mL/min
with UV detection at 254 nm. The purified products were
isolated as TFA salts and were converted to the corre-
sponding hydrochloride salts by addition of a solution of
hydrogen chloride (ꢀ1.25 M in methanol) followed by
evaporation; this process was repeated twice.
References and notes
1. Gellert, M.; Mizuuchi, K.; OÕDea, M. H.; Itoh, T.;
Tomizawa, J. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4772.
2. Sugino, A.; Peebles, C. L.; Kruezer, K. N.; Cozzarelli, N.
R. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4767.
3. Kato, J.; Nishimura, Y.; Imamura, R.; Niki, H.; Hiraga,
S.; Suzuki, H. Cell 1990, 63, 393.
21. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60, 7508.
22. MICs were determined by broth microdilution using
conditions recommended by the NCCLS (see National
Committee for Clinical Laboratory Standards. 2001.
Performance standards for antimicrobial susceptibility
4. Brighty, K. E.; Gootz, T. D. In The Quinolones; Andriole,
V. T., Ed., 3rd ed.; Academic: New York, 2000; pp 33–97.
5. Chu, D. T. W.; Fernandes, P. B. Antimicrob. Agents
Chemother. 1989, 33, 131.

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J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1272–1276
testing: 11th informational supplement. Vol. 21, no. 1,
24. Elsea, S. H.; McGuirk, P. R.; Gootz, T. D.; Moynihan,
M.; Osheroff, N. Antimicrob. Agents Chemother. 1993, 37,
2179.
25. The EC2 value is defined as the effective concentration of
drug required to enhance enzyme-mediated cleavage of
double-stranded DNA twofold.
M100-S11. National Committee for Clinical Laboratory
Standards, Wayne, PA). The MIC was defined as the
lowest concentration of each compound resulting in
inhibition of visible growth of bacteria after incubation
at 37 ꢁC for 18–24 h.
´
23. The CC50 value is defined as the concentration of
drug that is lethal to 50% of the cells. The Hep2
human laryngeal carcinoma cell line was incubated
with drug at 37 ꢁC for 72 h to generate eight-point
CC50 data.
26. Radl, S.; Dax, S. Curr. Med. Chem. 1994, 1, 262.
27. The IC50 value is defined as the concentration of drug
required to inhibit 50% of the supercoiling (decatenation)
of double-stranded DNA catalyzed by gyrase (topoiso-
merase IV).